Differential regulation of matrix metalloproteinase-2 and -9 expression and activity in adult rat cardiac fibroblasts in response to interleukin-1beta

Matrix metalloproteinases (MMPs), a family of endoproteinases, are implicated in cardiac remodeling. Interleukin-1beta (IL-1beta), which is increased in the heart following myocardial infarction, increases expression and activity of MMP-2 (gelatinase A) and -9 (gelatinase B) in cardiac fibroblasts. Previously, we have shown that IL-1beta activates ERK1/2, JNKs, and protein kinase C (PKC). However, signaling pathways involved in the regulation of MMP-2 and -9 expression and activity are not yet well understood. Using adult rat cardiac fibroblasts, we show that inhibition of ERK1/2 and JNKs inhibits IL-1beta-stimulated increases in MMP-9, not MMP-2, expression and activity. Chelerythrine, an inhibitor of PKC, inhibited activation of ERK1/2 and JNKs and expression and activity of both MMPs. Selective inhibition of PKC-alpha/beta1 using G?6976 inhibited JNKs activation and the expression and activity of MMP-9, not MMP-2. Inhibition of PKC-theta and PKC-zeta using pseudosubstrates inhibited IL-1beta-stimulated activation of ERK1/2 and JNKs and the expression and activity of MMP-2 and -9. Inhibition of PKC-epsilon had no effect. IL-1beta activated NF-kappaB pathway as measured by increased phosphorylation of IKKalpha/beta and IkappaB-alpha. Inhibition of ERK1/2, JNKs, and PKC-alpha/beta1 had no effect on NF-kappaB activation, whereas inhibition of PKC-theta and PKC-zeta inhibited IL-1beta-stimulated activation of NF-kappaB. SN50, NF-kappaB inhibitor peptide, inhibited IL-1beta-stimulated increases in MMP-2 and -9 expression and activity. These observations suggest that 1) activation of ERK1/2 and JNKs plays a critical role in the regulation of MMP-9, not MMP-2, expression and activity; 2) PKC-alpha/beta1 act upstream of JNKs, not ERK1/2; 3) PKC-zeta and -theta, not PKC-epsilon, act upstream of JNKs, ERK1/2, and NF-kappaB; and 4) activation of NF-kappaB stimulates expression and activity of MMP-2 and -9.     

ERK1/2 and JNKs, but not p38 kinase, are involved in reactive oxygen species-mediated induction of osteopontin gene expression by angiotensin II and interleukin-1beta in adult rat cardiac fibroblasts

Osteopontin (OPN), also called cytokine Eta-1, expressed in the myocardium co-incident with heart failure plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Angiotensin II (Ang II) and inflammatory cytokines are increased in the heart following MI. We studied the involvement of mitogen-activated protein kinases (ERK1/2, JNKs, p38 kinase) and reactive oxygen species (ROS) in Ang II- and cytokine-induced OPN gene expression in adult rat cardiac fibroblasts. Ang II alone increased OPN mRNA (3.3 +/- 0.3-folds; P < 0.05; n = 7), while interleukin-1beta (IL-1beta), tumor necrosis factor (TNF-alpha), and interferon-gamma (IFN-gamma) had no effect. A combination of Ang II with IL-1beta or TNF-alpha, not IFN-gamma, increased OPN mRNA more than Ang II alone. Nitric oxide donor, S-nitrosoacetylpenicillamine (SNAP), alone or in combination with Ang II had no effect. Diphenylene iodonium (DPI), inhibitor of NAD(P)H oxidase, and tiron, superoxide scavenger, inhibited Ang II- and Ang II+ IL-1beta-stimulated increases in OPN mRNA. Ang II activated ERK1/2 within 5 min of treatment, not JNKs. IL-1beta activated ERK1/2 and JNKs within 15 min of treatment. A combination of Ang II and IL-1beta activated ERK1/2 within 5 min of treatment. None of these stimuli activated p38 kinase. DPI almost completely inhibited Ang II + IL-1beta-stimulated activation of ERK1/2, while partially inhibiting JNKs. PD98059, ERK1/2 pathway inhibitor, and SP600125, JNKs inhibitor, partially inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. A combination of PD98059 and SP600125 almost completely inhibited Ang II + IL-1beta-stimulated increases in OPN mRNA. Thus, Ang II alone increases OPN expression, while IL-1beta and TNF-alpha act synergistically with Ang II to increase OPN mRNA possibly via NO independent mechanisms. The synergistic increase in OPN mRNA involves ROS-mediated activation of ERK1/2 and JNKs, not P38 kinase, pathways in cardiac fibroblasts.     

Lysophosphatidic acid enhances interleukin-13 gene expression and promoter activity in T cells

Airway smooth muscle cells (ASMC) are a source of inflammatory chemokines that may propagate airway inflammatory responses. We investigated the production of the CXC chemokine growth-related oncogene protein-alpha (GRO-alpha) from ASMC induced by cytokines and the role of MAPK and NF-kappaB pathways. ASMC were cultured from human airways, grown to confluence, and exposed to cytokines IL-1beta and TNF-alpha after growth arrest. GRO-alpha release, measured by ELISA, was increased by >50-fold after IL-1beta (0.1 ng/ml) or 5-fold after TNF-alpha (1 ng/ml) in a dose- and time-dependent manner. GRO-alpha release was not affected by the T helper type 2 cytokines IL-4, IL-10, and IL-13. IL-1beta and TNF-alpha also induced GRO-alpha mRNA expression. Supernatants from IL-1beta-stimulated ASMC were chemotactic for neutrophils; this effect was inhibited by anti-GRO-alpha blocking antibody. AS-602868, an inhibitor of IKK-2, and PD-98059, an inhibitor of ERK, inhibited GRO-alpha release and mRNA expression, whereas SP-600125, an inhibitor of JNK, reduced GRO-alpha release without effect on mRNA expression. SB-203580, an inhibitor of p38 MAPK, had no effect. AS-602868 but not PD-98059 or SP-600125 inhibited p65 DNA-binding induced by IL-1beta and TNF-alpha. By chromatin immunoprecipitation assay, IL-1beta and TNF-alpha enhanced p65 binding to the GRO-alpha promoter, which was inhibited by AS-602868. IL-1beta- and TNF-alpha-stimulated expression of GRO-alpha from ASMC is regulated by independent pathways involving NF-kappaB activation and ERK and JNK pathways. GRO-alpha released from ASMC participates in neutrophil chemotaxis.     

Osteopontin inhibits interleukin-1beta-stimulated increases in matrix metalloproteinase activity in adult rat cardiac fibroblasts: role of protein kinase C-zeta

We have shown that osteopontin (OPN), an extracellular matrix protein, plays an important role in post myocardial infarction (MI) remodeling by promoting collagen synthesis and accumulation. Interleukin-1beta (IL-1beta), increased in the heart following MI, increases matrix metalloproteinase (MMP) activity in cardiac fibroblasts in vitro. Here, we show that OPN alone has no effect on MMP activity or expression. However, it reduces IL-1beta-stimulated increases in MMP activity and expression in adult rat cardiac fibroblasts. Pretreatment with bovine serum albumin had no effect on MMP activity or protein content, whereas GRGDS (glycine-arginine-glycine-aspartic acid-serine)-pentapeptide (which interrupts binding of RGD-containing proteins to cell surface integrins) and monoclonal antibody m7E3 (a rat beta3 integrins antagonist) inhibited the effects of OPN. Inhibition of PKC using chelerythrine inhibited the activities of both MMP-2 and MMP-9. Stimulation of cells using IL-1beta increased phosphorylation and translocation of PKC to membrane fractions, which was inhibited by OPN. OPN inhibited IL-1beta-stimulated increases in translocation of PKC-zeta from cytosolic to membrane fractions. Furthermore, the levels of phospho-PKC-zeta were lower in the cytosolic fractions of OPN knock-out mice hearts as compared with wild type 6 days post-MI. Inhibition of PKC-zeta using PKC-zeta pseudosubstrate inhibited IL-1beta-stimulated increases in MMP-2 and MMP-9 activities. These observations suggest that OPN, acting via beta3 integrins, inhibits IL-1beta-stimulated increases in MMP-2 and MMP-9 activity, at least in part, via the involvement of PKC-zeta. Thus, OPN may play a key role in collagen deposition during myocardial remodeling following MI by modulating cytokine-stimulated MMP activity.     

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.     

Reactive oxygen species produced by NADPH oxidase, xanthine oxidase, and mitochondrial electron transport system mediate heat shock-induced MMP-1 and MMP-9 expression

In addition to ultraviolet radiation, human skin is also exposed to infrared radiation (IR) from natural sunlight. IR typically increases the skin temperature. This study examined whether or not heat shock-induced ROS stimulates MMPs in keratinocyte HaCaT cells. In HaCaT cells, heat shock was found to increase the intracellular ROS levels, including hydrogen peroxide and superoxide. The heat shock treatment induced MMP-1 and MMP-9, but not MMP-2, at the mRNA and protein levels. Moreover, heat shock caused the rapid activation of the three distinct MAPKs, ERK, JNK, and p38 kinase. The heat shock-induced expression of MMP-1 and MMP-9 was significantly suppressed by a pretreatment with the antioxidant NAC or catalase. On the other hand, SOD inhibited heat shock-induced activity of MMP-9 induction, but not MMP-1. A pretreatment with NAC or catalase, but not SOD, attenuated the phosphorylation of ERK, JNK, and p38 kinase by heat shock. The potential sites of ROS generation by heat shock along with its role in the heat shock-induced expression of MMP-1 and MMP-9 were next analyzed. These results indicate that heat shock-induced ROS is promoted via NADPH oxidase, xanthine oxidase, and mitochondria. Indeed, the NADPH oxidase and xanthine oxidase activities were increased by heat shock. Overall, the ROS produced by heat shock may play an important role in the heat shock-induced activation of MAPKs, which can induce MMP-1 and-9 expressions.     

Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway

Homocysteine (Hcy) induces matrix metalloproteinase (MMP)-9 in microvascular endothelial cells (MVECs). We hypothesized that the ERK1/2 signaling pathway is involved in Hcy-mediated MMP-9 expression. In cultured MVECs, Hcy induced activation of ERK, which was blocked by PD-98059 and U0126 (MEK inhibitors). Pretreatment with BAPTA-AM, staurosporine (PKC inhibitor), or Gö6976 (specific inhibitor for Ca²⁺-dependent PKC) abrogated ERK phosphorylation, suggesting the role of Ca²⁺ and Ca²⁺-dependent PKC in Hcy-induced ERK activation. ERK phosphorylation was suppressed by pertussis toxin (PTX), suggesting the involvement of G protein-coupled receptors (GPCRs) in initiating signal transduction by Hcy and leading to ERK activation. Pretreatment of MVECs with genistein, BAPTA-AM, or thapsigargin abrogated Hcy-induced ERK activation, suggesting the involvement of the PTK pathway in Hcy-induced ERK activation, which was mediated by intracellular Ca²⁺ pool depletion. ERK activation was attenuated by preincubation with N-acetylcysteine (NAC) and SOD, suggesting the role of oxidation in Hcy-induced ERK activation. Pretreatment with an ERK1/2 blocker (PD-98059), staurosporine, folate, or NAC modulated Hcy-induced MMP-9 activation as measured using zymography. Our results provide evidence that Hcy triggers the PTX-sensitive ERK1/2 signaling pathway, which is involved in the regulation of MMP-9 in MVECs. calcium signaling; protein kinase C; Src; G protein-coupled receptor; nonreceptor tyrosine kinase; protein G_i; protein G_q; protein tyrosine kinase 2; microvascular endothelial cell; cardiovascular remodeling     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Selected contribution: estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappaB activation in VSM cells from female rats.

Epidemiological studies have demonstrated that hormone replacement therapy with estrogen (E2) or E2 plus progesterone in postmenopausal women decreases the age-associated risk of cardiovascular disease by 30-50%. Treatment of vascular smooth muscle (VSM) cells with physiological concentrations of E2 has been shown to inhibit growth factor-stimulated cell proliferation. In this study, we tested the hypothesis that E2 inhibits the age-associated increase in VSM cell proliferation by inhibiting nuclear factor (NF)-kappaB pathway. We investigated the effects of E2 treatment and adenovirus-mediated estrogen receptor (ER)-alpha gene transfer on cell proliferation and NF-kappaB activation using VSM cells cultured from 3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstrate that VSM cell proliferation was significantly increased (P < 0.05) in aged compared with young adult female rats. Treatment of VSM cells with physiological concentrations of E2 inhibited VSM cell proliferation, and this inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects of E(2) on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER-alpha gene into VSM cells. Constitutive and interleukin (IL)-1beta-stimulated NF-kappaB activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats. E2 treatment of VSM cells from aged female rats inhibited both constitutive and IL-1beta-stimulated NF-kappaB activation. ER-alpha gene transfer into VSM cells from aged female rats further augmented the inhibitory effects of E2. In conclusion, our data demonstrate that constitutive and IL-1beta-stimulated NF-kappaB activation is increased in VSM cells from aged female rats due to loss of E2 and this can be restored back to normal levels by ER-alpha gene transfer and E2 treatment. In addition, increased NF-kappaB signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.     

Normoxic induction of the hypoxic-inducible factor-1 alpha by interleukin-1 beta involves the extracellular signal-regulated kinase 1/2 pathway in normal human cytotrophoblast cells

During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of alpha and beta subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1 alpha, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1 beta stimulated the expression of HIF-1 alpha protein. Meanwhile, IL-1 beta also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1 beta induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1 alpha protein expression and VEGF secretion by IL-1 beta. These data indicate that, in normal human cytotrophoblast cells, IL-1 beta induces HIF- 1 alpha-mediated VEGF secretion and that IL-1 beta-stimulated ERK1/2 activation may be involved in this process.     

An absolute role of the PKC-dependent NF-kappaB activation for induction of MMP-9 in hepatocellular carcinoma cells

Matrix metalloproteinases (MMPs) play an important role in inflammation, tumor cell invasion, and metastasis. We found that phorbol-12-myristate-13-acetate (PMA)-stimulated invasion of the hepatocellular carcinoma (HCC) SNU-387 and SNU-398 cells and that PMA induced the secretion of MMP-9 in the cells, but did not induce the secretion of MMP-2. The PMA-induced MMP-9 secretion was abolished by treatment of a pan-protein kinase C (PKC) inhibitor, GF109203X, and an inhibitor of NF-kappaB activation, sulfasalazine, and partly inhibited by treatment of inhibitors of ERK pathway, PD98059 and U0126. In addition, the PMA-stimulated activation of the MMP-9 promoter was completely inhibited by a mutation of the NF-kappaB site within the MMP-9 promoter, but not completely by mutations of two AP-1 sites. Moreover, the MMP-9 induction by HGF and TNF-alpha was also completely inhibited by GF109203X and sulfasalazine, but not by PD98059 and U0126. These data demonstrate that the PKC-dependent NF-kappaB activation is absolute for MMP-9 induction and that the PKC-dependent ERK activation devotes to increase the expression level of MMP-9, in HCC cells.     

Carbon monoxide inhibits IL-17-induced IL-6 production through the MAPK pathway in human pulmonary epithelial cells

Interleukin (IL)-17 is a proinflammatory cytokine that is produced by activated memory CD4 T cells, which regulates pulmonary neutrophil emigration by the induction of CXC chemokines and cytokines. IL-17 constitutes a potential target for pharmacotherapy against exaggerated neutrophil recruitment in airway diseases. As a cytoprotective and anti-inflammatory gaseous molecule, carbon monoxide (CO) may also regulate IL-17-induced inflammatory responses in pulmonary cells. Herein, we examine the production of cytokine IL-6 induced by IL-17 and the effect of CO on IL-17-induced IL-6 production in human pulmonary epithelial cell A549. We first show that IL-17 can induce A549 cells to release IL-6 and that CO can markedly inhibit IL-17-induced IL-6 production. IL-17 activated the ERK1/2 MAPK pathway but did not affect p38 and JNK MAPK pathways. CO exposure selectively attenuated IL-17-induced ERK1/ERK2 MAPK activation without significantly affecting either JNK or p38 MAPK activation. Furthermore, in the presence of U0126 and PD-98059, selective inhibitors of MEK1/2, IL-17-induced IL-6 production was significantly attenuated. We conclude that CO inhibits IL-17-stimulated inflammatory response via the ERK1/2-dependent pathway.